Polycomb Group (PcG) proteins are often overexpressed in breast cancer cells. These proteins assemble into polycomb repressive complexes (PRCs), which via histone posttranslational modifications (histone PTM) act as transcriptional repressors. The targets of PRCs are often tumor suppressors and differentiation-specific genes. PcG proteins are known to regulate breast cancer stem cell (BCSC) phenotype and resistance to chemotherapeutics. Chemopreventive agents and drugs that inhibit PcG proteins or the activities of PRCs are likely to help in breast cancer prevention and treatment by inhibiting BCSC phenotype. At present, very little is known about the pharmacological inhibitors or chemopreventive agents that target expression and/or function of PcG proteins. Based on our preliminary data, we hypothesize that non-steroidal anti-inflammatory drugs such as aspirin, inhibit the expression of PcG protein BMI1. BMI1 is often co-overexpressed with another PcG protein EZH2, which encodes a methyl transferase that is required for PRC2 function. The methyl transferase activity and expression of EZH2 is known to be inhibited by DZNep (3-Deazaneplanocin A) and other S- adenosyl-methionine (SAM) competitive inhibitors. Since BMI1 and EZH2 are both co-overexpressed and are required for full activity of PRCs, we further hypothesize that aspirin and DZNep may synergize to inhibit expression of PcG protein and histone PTM activity of PRCs, and oncogenic properties including BCSC phenotype. The specific aims of the proposal are- Aim 1: Examine the effect of aspirin and DZNep on breast cancer and breast cancer stem cells (BCSC). Aim 2: Determine whether aspirin and DZNep inhibits BCSC phenotype by regulating expression of polycomb group proteins. Aim 3: Examine whether aspirin alone and together with DZNep inhibits tumor initiating activity of breast cancer stem cells using mouse xenograft studies. These studies are designed to establish whether the PcG proteins are important targets of aspirin in cancer cells, and whether aspirin can be used to further potentiate anti-cancer activity of epigenome modifying drug DZNep. We hope these studies will provide a new paradigm for using aspirin and other NSAIDs together with epigenome modifying drugs such as DZNep to inhibit BCSC phenotype, breast cancer progression, metastasis and recurrence.